ABSTRACT Viral pathogens cause enormous pain and suffering, and more than 1B infections and 100M deaths each year. Unfortunately, safe and effective therapeutic interventions or vaccines are unavailable for many viral pathogens of global consequence. The identification of neutralizing antibodies (nAbs) against these pathogens would enable passive serotherapy as well as vaccine antigen (Ag) identification. However, determining the functional repertoire of nAbs elicited by viral infection faces two daunting challenges. First, several immunization- based methodologies enable antibody (Ab) discovery. These approaches reliably support the generation of polyclonal sera and, with hybridoma technology, renewable sources of monoclonal Abs. However, polyclonal Abs are non-renewable and hybridoma generation is costly, time-consuming, and results in a substantial loss of Ab diversity (i.e., biased). Direct screening of the vast repertoire of primary B cells is ideal for nAb discovery. However, primary plasmablast cultivation is challenging, and the limited lifespan of Ab- producing primary plasmablasts in culture makes direct screening of these cells impractical. Second, available assays for testing Ab function are low throughput, costly, and require enormous commitments of manpower and resources to execute. Here, an innovative method that can address all of these challenges is proposed. Heterologous Ab secretion by yeast cells that can be readily cultivated, and do not perturb viral infection of host cells, constitute an attractive alternative. This proposal will develop FAST (Functional Ab Screening Technology), a novel droplet microfluidics platform that combines high-throughput microfluidics and yeast Ab secretion library technology to rapidly identify nAbs against viruses, achieving dramatic increases in throughput and corresponding reductions in cost, compared to conventional systems. Importantly, because the biological functions of Abs expressed in yeast (viral neutralization) are directly tested in the system rather than simple Ag-Ab binding interactions, FAST is expected to return high-quality, biologically meaningful hits. To date, exciting proof-of concept data showing that the FAST system can be used to identify Abs that neutralize murine hepatitis virus (MHV) infection has been collected. To test the central hypothesis that FAST will deliver a rapid, low cost, unbiased, approach to identifying Abs that neutralize viral infection, this proposal will: AIM 1. To optimize FAST by executing a mock screen using a model system against MHV; AIM 2. To test the working hypothesis that an Ab library derived from MHV-infected mice contains potent MHV neutralizing molecules. If successful, this work will establish the first high-throughput system for performing functional viral neutralization bioassays at single-cell (digital) resolution, allowing identification of nAbs against viral pathogens of global consequence.